The importance of deoxycytidine kinase (dCK) has been emphasized by the findings that several clinically useful antitumor and antiviral compounds require this enzyme exclusively for the initial, rate-limiting step in their activation to therapeutic forms. Previously we have demonstrated than UTP is the true physiologic phosphate donor for dCK, and that inhibition of dCK activity by dCTP is dramatically different in the presence of UTP compared to ATP. We now demonstrate that these results are due to previously unrecognized allosteric effects of UTP on dCK. The investigations proposed in this application will extend these results by defining the biochemical and molecular characteristics of this regulation of dCK by UTP. The overall goal of these studies is to optimize the activity of dCK in intact cells to increase the activation and thus the cytotoxicity of therapeutic nucleoside analog substrates. First, we will use highly purified dCK from MOLT-4 T lymphoblasts to define the mechanistic differences observed with UTP compared to ATP by determining the number of substrate binding sites, measure the dissociation constants for dCyd at the nucleoside-binding site as well as the for UTP and ATP at the allosteric site, and determine the mechanism by which high concentrations of dCyd inhibit dCK in the presence of UTP. Second, we will define the allosteric binding site for UTP through a biochemical comparison of this enzyme from MOLT-4 cells and an E. coli expression vector which lacks the modification necessary for UTP to act as an allosteric effector. In addition, we will utilize site-directed mutagenesis to further identify the relevant molecular sites for enzyme activity. Third, we will elucidate the mechanism by which fluoroadenine arabinoside 5'-triphosphate increases phosphorylation of cytosine arabinoside (araC) by dCK, determine whether the phosphorylation of other analogs can also be enhanced and evaluate other nucleotides as stimulators of dCK. Furthermore, we will use intact cells displaying varying levels of dCK protein and sensitivity to araC to determine whether fluoroadenine arabinoside can enhance activation of araC in cells relatively resistant to the cytotoxic effects of this drug. Fourth, we will determine whether the 5'-mono or triphosphate derivatives of nucleoside analog substrates for dCK can inhibit phosphorylation of the parent compounds. These results will provide new information that will aid in the discovery of new substrates and stimulators for dCK. In addition, these results will assist in the design of protocols to enhance the therapeutic activity of clinically important nucleoside analogs such as araC and dFdC.